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Abstract:

Formulations of VLA-4 binding antibody are described.

Claims:

1-34. (canceled)

35. A method of treatment of a patient with an inflammatory disorder
comprising subcutaneous or intramuscular administration of a composition
comprising a VLA-4 binding antibody.

36. The method of claim 35, wherein the inflammatory disorder is multiple
sclerosis, asthma, an arthritic disorder, diabetes, a fibrotic disorder,
or an inflammatory bowel disease.

37. The method of claim 35, wherein the administration is subcutaneous.

38. The method of claim 35, wherein the VLA-4 binding antibody is
natalizumab or a variant of natalizumab, wherein said variant has light
and heavy chain variable region sequences, and wherein either the light
chain variable region sequence or the heavy chain variable region
sequence or both the light and heavy chain variable region sequences of
said variant differ from the corresponding sequence or sequences of
natalizumab by at least one amino acid, and wherein the light chain
variable region sequence of said variant differs from the light chain
variable region of natalizumab by not more than 6 amino acid residues and
the heavy chain variable region sequence of said variant differs from the
heavy chain variable region of natalizumab not more than 6 amino acid
residues.

39. The method of claim 35, wherein the composition comprises 120-190
mg/mL of the antibody.

40. The method of claim 35, wherein the composition comprises 140-160
mg/mL of the antibody.

41. The method of claim 35, wherein the composition comprises about 150
mg/mL of the antibody.

42. The method of claim 35, wherein the composition comprises 5-30 mM of
sodium phosphate buffer and the pH of the composition is about 6.+-.0.5.

44. The method of claim 35, wherein the composition comprises about 0.01%
(w/v) to about 0.1% (w/v) of polysorbate 80.

45. The method of claim 35, wherein the composition comprises about 0.4%
w/v of polysorbate 80.

46. The method of claim 35, wherein the composition has a dynamic
viscosity of 5-30 cP at ambient temperature.

47. The method of claim 35, wherein the composition comprises a salt at a
concentration of 100-200 mM.

48. The method of claim 47, wherein the composition comprises sodium
chloride at a concentration of about 140 mM.

49. The method of claim 35, wherein the composition is stable for at
least 12 months at a temperature of about 2.degree. C. to about 8.degree.
C.

50. A pre-filled syringe comprising a unit dose of about 1 ml of an
aqueous composition comprising 135 mg to 165 mg of natalizumab for
subcutaneous delivery.

51. A method of preparing an aqueous composition comprising about 120 to
190 mg/mL natalizumab and polysorbate in a phosphate buffer comprising:
(i) expressing the natalizumab in cell culture, (ii) passing the
natalizumab through at least one chromatography purification step, (iii)
passing the antibody through at least two ultrafiltration/diafiltration
steps in phosphate buffer, (iv) passing the natalizumab through at least
one ultrafiltration step in phosphate buffer, and (v) adjusting the
concentration of the natalizumab to about 120 mg/mL to 190 mg/mL.

52. The method of claim 51, wherein the composition further comprises
about 140 mM of sodium chloride.

53. The method of claim 51, wherein the composition comprises about 0.4%
w/v of polysorbate 80.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. application Ser. No.
60/944,076, filed Jun. 14, 2007, the entire contents of which are hereby
incorporated by reference.

BACKGROUND

[0002] Multiple sclerosis (MS) is one of the most common diseases of the
central nervous system. Today over 2,500,000 people around the world have
MS.

SUMMARY

[0003] The invention is based, in part, on the development of formulations
containing high concentrations of VLA-4 binding antibody. Some
embodiments are suitable for delivery to a subject, such as a human,
e.g., a human patient, by subcutaneous (SC) or intramuscular (IM)
delivery. The formulations are also suitable for intravenous (IV)
administration, e.g., when diluted into an acceptable infusion matrix
(such as normal saline). The VLA-4 binding antibody can be natalizumab,
for example, and the antibody concentration ranges from about 120 mg/mL
to about 190 mg/mL. The formulations provide a therapeutic effect for an
inflammatory, immune, or autoimmune disorder. For example, the
formulation can provide a therapeutic effect for a central nervous system
(CNS) inflammatory disorder, such as multiple sclerosis (MS).

[0004] In one aspect, the invention features an aqueous pharmaceutical
composition, such as a stable aqueous pharmaceutical composition,
containing a VLA-4 binding antibody at a concentration of about 120 to
about 190 mg/mL (e.g., at a concentration of about 135 mg/mL, about 140
mg/mL, about 150 mg/mL, about 160 mg/mL, or about 165 mg/mL), and a
phosphate buffer having about pH 5.5 to about pH 6.5. In some
embodiments, the VLA-4 antibody concentration is from about 130 mg/mL to
about 180 mg/mL or about 140 mg/mL to about 160 mg/mL. In one embodiment,
the VLA-4 antibody concentration is greater than about 150 mg/mL, e.g.,
it is in a range of greater than about 150 mg/mL to about 190 mg/mL. In
one embodiment, the VLA-4 antibody concentration is about 150 mg/mL.

[0005] In one embodiment, the VLA-4 binding antibody is a humanized
monoclonal antibody, such as natalizumab. In another embodiment, the
VLA-4 binding antibody is a variant of natalizumab. For example, in some
embodiments, the light chain variable region of the antibody has an amino
acid sequence that differs by one or more amino acid residues, but not
more than 2, 3, 4, 5, or 6 amino acid residues of the light chain
variable region of natalizumab, and/or the heavy chain variable region
has an amino acid sequence that differs by one or more amino acid
residues, but not more than 2, 3, 4, 5, or 6 amino acid residues of the
heavy chain variable region of natalizumab. In some embodiments, some or
all differences are conservative changes.

[0006] In another embodiment, the VLA-4 binding antibody has one or both
of a light chain variable region having the amino acid sequence of SEQ ID
NO:7 in U.S. Pat. No. 5,840,299, which is incorporate by reference
herein, and a heavy chain variable region having the amino acid sequence
of SEQ ID NO:11 in U.S. Pat. No. 5,840,299. In other embodiments, the
VLA-4 antibody is a variant of one of these antibodies. For example, in
some embodiments, the light chain variable region has an amino acid
sequence that differs by one or more amino acid residues, but not more
than 2, 3, 4, 5, or 6 amino acid residues from the sequence in SEQ ID
NO:7 in U.S. Pat. No. 5,840,299, and/or the heavy chain variable region
has an amino acid sequence that differs by one or more amino acid
residues, but not more than 2, 3, 4, 5, or 6 amino acid residues as
defined by SEQ ID NO:11 in U.S. Pat. No. 5,840,299.

[0007] In yet another embodiment, the VLA-4 binding antibody has one or
both of a light chain amino acid sequence of SEQ ID NO:1 in Table 1-1,
and a heavy chain amino acid sequence of SEQ ID NO:2 in Table 1-2. In
other embodiments, the VLA-4 antibody is a variant of one of these
antibodies. For example, in some embodiments, the light chain of the
antibody has an amino acid sequence that differs by one or more amino
acid residues, but not more than 2, 3, 4, 5, or 6 amino acid residues
from the sequence of SEQ ID NO:1, and/or the heavy chain of the antibody
has an amino acid sequence that differs by one or more amino acid
residues, but not more than 2, 3, 4, 5, or 6 amino acid residues from the
sequence of SEQ ID NO:2.

[0008] A "difference" in amino acid sequence, as used in this context,
means a difference in the identity of an amino acid (e.g., a substitution
of a different amino acid for an amino acid in SEQ ID NO:7 or 11 referred
to above) or a deletion or insertion. A difference can be, for example,
in a framework region, a CDR, a hinge, or a constant region. A difference
can be internal or at the end of a sequence of protein. In some
embodiments, some or all differences are conservative changes as compared
to the recited sequence.

[0009] In certain embodiments, the pH of the composition is about
6.0±0.5 (e.g., about 5.0±0.5, about 6.0±0.5, about 7.0±0.5),
and the phosphate buffer composition is between about 5 mM and about 30
mM (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM). In another
embodiment, the composition further comprises a salt, such as sodium
chloride, at a concentration of between about 100 mM and about 200 mM
(e.g., about 120 mM, 140 mM, 160 mM, 180 mM). In another embodiment, the
composition comprises L-arginine hydrochloride, or glycerol. In another
embodiment, the composition contains an amino acid, such as glycine, at a
concentration of about 200 mM to about 300 mM (e.g., about 220 mM, 240
mM, 260 mM, 280 mM). In another embodiment, the composition contains a
pharmaceutically acceptable excipient, such as a surfactant, such as
polysorbate 80, in an amount of about 0.001% to about 2.0%, about 0.004%
to about 0.4%, about 0.008 to about 0.2%, about 0.02% to about 0.08%
(w/v) (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about
0.05%, about 0.06%, about 0.07%, about 1%, about 1.5%).

[0010] In certain embodiments, the composition includes glycerol, and
contains substantially no L-arginine hydrochloride, or sodium chloride.
In other embodiments, the composition includes L-arginine hydrochloride,
but substantially no glycerol or sodium chloride (other than that from
the phosphate buffer and the L-arginine hydrochloride). In other
embodiments, the composition includes sodium chloride, but substantially
no glycerol or L-arginine hydrochloride.

[0011] In some embodiments, the antibody formulation includes a histidine
buffer, e.g., instead of a phosphate buffer, and the histidine buffer is
about pH 5 to about pH 7 (e.g., about pH 5.5±0.5, pH 6±0.5, or pH
6.5±0.5). The histidine buffer composition is between about 10 mM and
about 30 mM (e.g., about 15 mM, about 20 mM, about 25 mM). The histidine
buffer formulation also includes about 200 mM to about 300 mM glycerol
(e.g., about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280
mM glycerol), and polysorbate 80 to about 0.001% to about 2.0% (w/v)
(e.g., about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%,
about 0.07%, about 1%, about 1.5%). The histidine formulation optionally
includes about 5 mM to about 15 mM L-methionine (e.g., about 10 mM
L-methionine).

[0013] In one embodiment, the composition featured herein is a liquid. In
another embodiment, the composition is stable for at least 12 months
(e.g., at least 24, 30, 36 months), at a temperature of about 2°
C. to about 8° C. (e.g., about 5° C.). In another
embodiment, the composition is stable for at least 2, 3, 4, 5, 6, or 7
days (e.g., at least one week or 12 or 14 days). at ambient temperature
(about 20-30° C., such as about 25° C.).

[0014] In yet another embodiment, the composition is suitable for SC or IM
administration. In even another embodiment, the composition is suitable
for IV administration.

[0015] In another aspect, the invention features a method of preparing an
aqueous composition, such as a stable aqueous composition, that includes
about 120 to about 190 mg/mL VLA-4 binding antibody and polysorbate in a
phosphate buffer. The method includes expressing the antibody in cell
culture, passing the antibody through at least one chromatography
purification step, passing the antibody through at least two
ultrafiltration/diafiltration steps in phosphate buffer, passing the
antibody through at least one ultrafiltration step in phosphate buffer,
and adjusting the concentration of the antibody, e.g., downward, to about
120 mg/mL to about 190 mg/mL, by adding polysorbate and/or phosphate
buffer. In one embodiment, the VLA-4 binding antibody is natalizumab, and
in another embodiment the polysorbate is polysorbate 80. The
concentration of the antibody can be, e.g., about 135 mg/mL to about 165
mg/mL, e.g., about 150 mg/mL. In some embodiments, the phosphate buffer
includes other excipients such as glycerol, L-arginine hydrochloride, or
sodium chloride. The final formulation has a pH of about 5 to about 7,
e.g., from about 5.5 to about 6.5.

[0016] In another aspect, the invention features a delivery device
designed for or suitable for SC or IM administration, where the delivery
device is packaged with or contains a unit dose of a composition
described herein, e.g., a composition containing a concentrated
formulation of natalizumab suitable for SC or IM administration. In one
embodiment, the unit dose is about 100 mg to about 450 mg (e.g., about
120 mg to about 350 mg; about 150 mg, about 200 mg, about 250 mg, about
300 mg). In one embodiment, the unit dose ranges from greater than about
100 mg to about 450 mg. In another embodiment, the unit dose will deliver
between about 1.4 mg/kg and about 3.0 mg/kg VLA-4 binding antibody or
fragment thereof per kg of body weight to the human. In another
embodiment, the unit dose is about 0.25 mL to about 1.5 mL (e.g., about
0.5 mL, about 0.75 mL, about 1.0 mL).

[0017] In one embodiment, a unit dose is about 300 mg natalizumab, and in
another embodiment, the unit dose is divided into fractions, such as into
two halves, each half containing about 150 mg of a VLA-4 binding
antibody. In yet another embodiment, a patient is administered
natalizumab as a regimen. In one embodiment, the patient is administered
about 300 mg natalizumab once per month, e.g., by the administration of
two sequential doses of 150 mg natalizumab. In an alternative embodiment,
the patient is administered about 300 mg natalizumab per month,
administered by a first dose of 150 mg natalizumab, then a second dose of
150 mg natalizumab about two weeks later.

[0018] The invention features methods that optimize provision of a highly
concentrated liquid formulation of a VLA-4 binding antibody, e.g.,
natalizumab, to a patient.

[0019] In one embodiment, the method allows for a gradual increase in the
concentration of the antibody provided. This allows ramp-up of antibody
concentration and can allow monitoring of the patient for tolerance,
reactions and the like as the concentration is increased. For example,
the method can start by providing natalizumab to the patient at one or
more initial or relatively low concentrations followed by providing
natalizumab to the patient at a final, higher concentration. Exemplary
formulations for the initial concentration will typically have an
antibody concentration of less than 80%, 70%, 50%, 30%, 20% or 10% of the
final higher concentration. Typical initial concentrations can be, e.g.,
20 mg/mL, 30 mg/mL, or 40 mg/mL. Typical final concentrations will be,
e.g., about 120 mg/mL to about 190 mg/mL (e.g., about 135 mg/mL, about
140 mg/mL, about 150 mg/mL, about 160 mg/mL, or about 165 mg/mL). In some
embodiments, the patient will receive one, or a plurality of
administrations at one or a plurality of initial concentrations. For
example, in one embodiment, the patient will receive increasing
concentrations over a number of administrations. In some embodiments, the
patient will receive 2, 3, 4, 5, 6, 7, or 8 administrations at one or
more initial concentrations prior to reaching the final concentration.
For example, the patient will receive one or more administrations at a
first initial concentration, and one or more administrations at a second
higher concentration. In some embodiments, the patient is assessed after
one or more administrations for symptoms, including adverse symptoms. In
some embodiments, the patient is administered a formulation having an
increased concentration of natalizumab only after determining that the
patient does not have an unacceptable adverse reaction to the previous
administration.

[0020] In one embodiment, the method allows for a gradual increase in the
antibody dosage provided (dosage as used here refers to the amount of
antibody provided in one, or in each of a defined small number, e.g., 2,
administrations). This allows ramp-up of dosage and can allow monitoring
of the patient for tolerance, adverse reactions, and the like as the
dosage is increased. For example, the method can begin by providing
natalizumab to the patient at one or more initial or relatively low
dosages followed by providing natalizumab to the patient at a final,
higher dosage. Typical initial dosages can be, e.g., 80%, 70%, 50%, 30%,
20% or 10% or less of the final higher dosage. Typical final dosages will
vary based on the frequency of administration once steady state
administration has been achieved. For example, some embodiments include
final dosages of between 75 mg and 500 mg (e.g., 100 mg, 150 mg, 200 mg,
250 mg, 300 mg, 350 mg, 400 mg, 450 mg) (these dosages can be typical of
approximately monthly administration). Other embodiments include final
dosages of between 50 mg and 250 mg (e.g., 75 mg, 100 mg, 150 mg, 200 mg)
(these dosages are typical of administration every two weeks). Other
embodiments include final dosages of between 25 mg and 150 mg (e.g., 50
mg, 75 mg, 100 mg, 125 mg) (these dosages are typical of weekly
administration). In some embodiments, the patient will receive one or a
plurality of administrations, at one or a plurality of initial dosages.
For example, in one embodiment, the patient will receive increasing
dosages over a number of administrations. In some embodiments, the
patient will receive 2, 3, 4, 5, 6, 7, or 8 administrations at one or
more initial dosages prior to reaching the final dosage. For example, the
patient will receive one or more administrations at a first initial
dosage, and one or more administrations at a second higher initial
dosage. In some embodiments, the patient is assessed after one or more
administrations for symptoms, including adverse symptoms. In some
embodiments, the patient is administered an increased dosage of
natalizumab only after determining that the patient does not have an
unacceptable adverse reaction to the previous dosage.

[0021] The invention also includes kits, e.g., starter packs, for
implementing a ramp-up of concentration or dosage. In one embodiment, the
patient, or a healthcare provider, is provided with a kit or "starter
pack" of natalizumab formulations, including packages of increasing
concentrations or dosages of natalizumab. The patient or healthcare
provider provided with a starter pack is instructed to self-administer or
administer a first, e.g., a low, or the lowest dosage or concentration of
natalizumab, and to wait a designated period time. If the patient
experiences no, or a minor level of, adverse symptoms, the patient or
health care provider is instructed to self-administer or administer a
second formulation, e.g., a higher, e.g., the next highest concentration
or dosage. The patient or healthcare provider is instructed to continue
the step-wise increase in dosages or concentrations until the desired
dosage or concentration is achieved. The patient or healthcare provider
may be instructed to maintain self-administration or administration of
the final formulation at regular intervals for a specified period of
time.

[0022] In one embodiment, the highly concentrated formulation of VLA-4
binding antibody is provided to a patient prepacked in a suitable
delivery device, such as a syringe.

[0023] In another aspect, the invention features a method, e.g., a method
of instructing a patient in need of a VLA-4 binding antibody therapy, how
to administer a formulation described herein. The method includes (i)
providing the patient with at least one unit dose of a highly
concentrated formulation of VLA-4 binding antibody described herein; and
(ii) instructing the patient to self-administer the at least one unit
dose intramuscularly or subcutaneously. Another method, e.g., a method of
treatment, includes (i) providing the patient with at least two unit
doses of a highly concentrated formulation of VLA-4 binding antibody; and
(ii) instructing the patient to self-administer the unit doses
subcutaneously or intramuscularly, e.g., one dose at a time.

[0025] Another aspect, the invention features a unit dose of a
concentrated formulation of VLA-4 binding antibody described herein,
where the unit dose is about 0.25 mL to about 1.5 mL (e.g., about 0.5 mL,
about 0.75 mL, or about 1.0 mL). In one embodiment, a unit dose is about
100 mg to about 450 mg (e.g., about 150 mg, about 160 mg, about 180 mg,
about 200 mg, about 250 mg, about 300 mg, or about 350 mg).

[0026] In another aspect, the invention features a unit dose of an aqueous
formulation of VLA-4 binding antibody, where administration of the unit
dose to a human will deliver between about 1.4 mg and about 3.0 mg VLA-4
binding antibody or fragment thereof per kg of body weight to the human.

[0027] In another aspect, the invention features a method of treating a
patient by administering to the patient a composition containing a VLA-4
binding antibody in a formulation suitable for SC or IM administration.
In one embodiment, the patient has an inflammatory disorder, such as
multiple sclerosis, asthma, rheumatoid arthritis, diabetes, or Crohn's
disease. In another embodiment, the composition is administered as a
regimen. In another embodiment, the method further includes selecting a
patient suitable for treatment with the composition. A patient suitable
for treatment, for example, has demonstrated a sign or symptom indicative
of disease onset, such as a sign or symptom indicative of MS. In yet
another embodiment, the method further includes administering to the
patient a second therapeutic agent, such as, a thrombolytic agent, a
neuroprotective agent, an anti-inflammatory agent, a steroid, a cytokine,
or a growth factor.

[0028] In another aspect, the invention features a method of evaluating a
patient by determining if the patient meets a preselected criterion, and
if the patient meets the preselected criterion approving, providing,
prescribing, or administering a VLA-4 binding antibody formulation
described herein to the patient. In one embodiment, the preselected
criterion is the failure of the patient to adequately respond to a prior
alternate therapeutic treatment or regimen, e.g., for treatment of MS. In
another embodiment, the preselected criterion is the absence of any signs
or symptoms of progressive multifocal leukoencephalopathy (PML), or the
absence of any diagnosis of PML. In another embodiment, the criterion is
as described in U.S. Ser. No. 60/836,530, filed Aug. 9, 2006, hereby
incorporated by reference, which describes methods and systems for drug
distribution.

[0029] In another aspect, the invention features a method of instructing a
recipient on the administration of a highly concentrated formulation of
natalizumab. The method includes instructing the recipient (e.g., an end
user, patient, physician, retail or wholesale pharmacy, distributor, or
pharmacy department at a hospital, nursing home clinic or HMO) that the
drug should be administered to a patient subcutaneously or
intramuscularly.

[0030] In another aspect, a method of distributing a composition described
herein is provided. The composition contains a highly concentrated
formulation of natalizumab and is suitable for subcutaneous or
intramuscular or intravenous administration. The method includes
providing a recipient (e.g., an end user, patient, physician, retail or
wholesale pharmacy, distributor, or pharmacy department at a hospital,
nursing home clinic or HMO) with a package containing sufficient unit
dosages of the drug to treat a patient for at least 6, 12, 24, or 36
months.

[0031] In another aspect, the invention features a method of evaluating
the quality of a package or lot of packages (e.g., to determine if it has
expired) of a composition described herein containing a highly
concentrated amount of VLA-4 binding antibody. The method includes
evaluating whether the package has expired. The expiration date is at
least 6, 12, 24, 36, or 48 months, e.g., greater than 24 or 36 months,
from a preselected event, such as manufacturing, assaying, or packaging.
In some embodiments, a decision or step is taken as a result of the
analysis, e.g., the antibody in the package is used or discarded,
classified, selected, released or withheld, shipped, moved to a new
location, released into commerce, sold, or offered for sale, withdrawn
from commerce or no longer offered for sale, depending on whether the
product has expired.

[0032] In another aspect, the invention features a package containing at
least 2 unit doses of an aqueous composition containing a highly
concentrated amount of VLA-4 binding antibody. In one embodiment, all of
the unit doses contain the same amount of antibody, and in other
embodiments, there are unit dosages of two or more strengths, or two or
more different formulations, e.g., having different strengths or release
properties). In one embodiment, at least one dosage contains about 100 mg
to about 450 mg of VLA-4 binding antibody, e.g., about 100 mg, about 200
mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg of VLA-4
binding antibody.

[0033] In another aspect, the invention includes a method of instructing a
recipient on the administration of an aqueous formulation containing
VLA-4 binding antibody. The method includes instructing the recipient
(e.g., an end user, patient, physician, retail or wholesale pharmacy,
distributor, or pharmacy department at a hospital, nursing home clinic or
HMO) that the antibody should be administered to a patient prior to the
expiration date. The expiration date is at least 6, 12, 18, 24, 36, or 48
months, e.g., greater than 18, 24 or 36 months, from a preselected event,
e.g., manufacturing, assaying, or packaging. In one embodiment, the
recipient also receives a supply of the antibody, e.g., a supply of unit
dosages.

[0034] In another aspect, the invention features the use of a method or
system described in PCT/US2007/075577 (published as WO/2008/021954) with
a formulation described herein. Embodiments include a method of
distributing a formulation described herein, monitoring or tracking the
provision of a formulation described herein to a pharmacy, infusion
center, or patient, monitoring one or more patients, selecting patients,
or compiling or reporting data on the use of a formulation described
herein. PCT/US2007/075577 (published as WO/2008/021954) is hereby
incorporated by reference.

[0035] In another aspect, the invention features a method of selecting a
patient for treatment with a formulation described herein for a disorder
described herein, e.g., multiple sclerosis. The method includes:

[0036] selecting or providing a patient who has been treated by
intravenous delivery of a VLA-4 binding antibody, e.g., natalizumab; and

[0037] providing or administering a formulation described herein to the
patient,

[0038] thereby treating the patient.

[0039] In another aspect, the invention features a method of analyzing a
product or a process, e.g., a manufacturing process. The method includes
providing an aqueous formulation of a highly concentrated VLA-4 binding
antibody composition, e.g., one made by a process described herein, and
providing an evaluation of the formulation by assessing a solution
parameter, such as color (e.g., colorless to slightly yellow, or
colorless to yellow), clarity (e.g., clear to slightly opalescent or
clear to opalescent), or viscosity (e.g., between approximately 5 cP and
30 cP (e.g., 10 cP, 20 cP) when measured at ambient temperature, such as
at 20° C.-30° C., e.g., 25° C.). The evaluation can
include an assessment of one or more solution parameters. Optionally, a
determination of whether the solution parameter meets a preselected
criteria is determined, e.g., whether the preselected criteria is
present, or is present in a preselected range, is determined, thereby
analyzing the process.

[0040] In one embodiment, evaluation of the process includes a measure of
the stability of the anti-VLA-4 antibody formulation. Stability of the
antibody formulation can be measured, for example, by aggregate
formation, which is assayed, e.g., by size exclusion high pressure liquid
chromatography (HPLC), by color, clarity, or viscosity as described
herein. A formulation can be determined to be stable, and therefore
acceptable for further processing or distribution, if the change in an
assay parameter is less than about 10%, 5%, 3%, 2%, 1%, 0.5%, 0.05%, or
0.005% or less, over a pre-set period of time, and optionally at a given
temperature. In one embodiment, a highly concentrated liquid anti-VLA-4
antibody formulation is stable for 1, 2, 3, 4, or 5 days or more at room
temperature (e.g., at about 18° C., 19° C., 20° C.,
21° C., 22° C., 23° C., 24° C., or 25°
C.).

[0041] In one embodiment, the method further includes comparing the value
determined with a reference value, to thereby analyze the manufacturing
process.

[0042] In one embodiment, the method further includes maintaining the
manufacturing process based, at least in part, upon the analysis. In one
embodiment, the method further includes altering the manufacturing
process based upon the analysis.

[0043] In another embodiment the method includes evaluating a process,
e.g., manufacturing process, of an aqueous formulation of highly
concentrated VLA-4 binding antibody made by a selected process, that
includes making a determination about the process based upon a method or
analysis described herein. In one embodiment, the method further includes
maintaining or altering the manufacturing process based, at least in
part, upon the method or analysis. Thus, in another embodiment the party
making the evaluation does not practice the method or analysis described
herein but merely relies on results which are obtained by a method or
analysis described herein.

[0044] In another embodiment the method includes comparing two or more
preparations in a method of monitoring or controlling batch-to-batch
variation or to compare a preparation to a reference standard.

[0045] In yet another embodiment, the method can further include making a
decision, e.g., to classify, select, accept or discard, release or
withhold, process into a drug product, ship, move to a different
location, formulate, label, package, release into commerce, sell or offer
for sale the preparation, based, at least in part, upon the
determination.

[0046] In another aspect, the invention features a method of storing,
distributing, or using a VLA-4 binding antibody formulation, e.g., a
natalizumab formulation, described herein. The method includes:

[0047] storing the formulation for a first period at a first, low
temperature, e.g., less than 18° C., e.g., from above freezing but
at or below 15° C., 10° C., or 4° C.;

[0048] storing the formulation for a second period at a second, higher
temperature, e.g., without refrigeration or at room temperature, e.g.,
between 18° C. and 25° C., wherein said second period is no
more than 24, 48, 72, or 96 hours, and where in some embodiments, the
second period ends upon administration to the patient or discard of the
formulation.

[0049] In another aspect, the invention features a method of storing,
distributing, or using a VLA-4 binding antibody formulation, e.g., a
natalizumab formulation, described herein. The method includes:

[0050] storing the formulation at a first, low temperature, e.g., less
than 18° C., e.g., from above freezing, but at or below 15°
C., 10° C., or 4° C.;

[0051] providing the formulation to a recipient, e.g., an end-user, e.g.,
a patient or healthcare provider;

[0052] optionally, instructing the end-user that the formulation can be
stored at a second, higher temperature, e.g., without refrigeration or at
room temperature, e.g., between 18° C. and 25° C.; and

[0053] after receipt by the recipient, storing the formulation for up to
24, 48, 72, or 96 hours at the second temperature.

[0054] In another aspect, the invention features a method of instructing
an entity, e.g., a pharmacy, distributor, or end-user, e.g., a patient or
healthcare provider, how to store, distribute, or use a VLA-4 binding
antibody formulation, e.g., a natalizumab formulation, described herein.
The method includes:

[0055] instructing the entity that the formulation should be stored at a
first, low temperature, e.g., less than 18° C., e.g., from above
freezing but at or below 15° C., 10° C., or 4° C.,
for a first period, where said first period extends up until the
formulation is provided to an end-user or until within 24, 48, 72, or 96
hours prior to administration to a patient; and

[0056] instructing the entity that the formulation can be stored at a
second, higher temperature, e.g., without refrigeration or at room
temperature, e.g., between 18° C. and 25° C. for a second
period, where said second period does not exceed 24, 48, 72, or 96 hours,
thereby instructing an entity.

[0057] In another aspect, the invention features a method of storing,
distributing, or using a VLA-4 binding antibody formulation, e.g., a
natalizumab formulation, described herein. The method includes:

[0058] storing the formulation at a first, low temperature, e.g., less
than 18° C., e.g., from above freezing but at or below 15°
C., 10° C., or 4° C.; and

[0059] storing the formulation at a second, higher temperature, e.g.,
without refrigeration or at room temperature, e.g., between 18° C.
and 25° C. for no more than 24, 48, 72, or 96 hours.

[0060] In another aspect, the invention features a method of evaluating,
such as evaluating the quality of, an aqueous formulation of highly
concentrated VLA-4 binding antibody, e.g., in a quality control or
release specification analysis. The method includes providing an
evaluation of an antibody formulation for a solution parameter, such as
color (e.g., colorless to slightly yellow, or colorless to yellow),
clarity (e.g., clear to slightly opalescent or clear to opalescent), or
viscosity (e.g., between approximately 5 cP and 30 cP when measured at
ambient temperature, such as at 20° C-30° C., e.g.,
25° C.). The evaluation can include an assessment of one or more
of the above parameters. The method also includes, optionally,
determining whether the solution parameter meets a preselected criteria,
e.g., whether the preselected criteria is present, or is present in a
preselected range. If the observed solution parameter is within a
preselected range of values, or meets the preselected standard criteria,
then the preparation is selected, such as for packaging, use, sale,
release into commerce, discarding etc.

[0061] In another aspect, the invention features a method of complying
with a regulatory requirement, e.g., a post approval requirement of a
regulatory agency, e.g., the FDA. The method includes providing an
evaluation of an antibody formulation for a solution parameter, such as
color (e.g., colorless to slightly yellow, or colorless to yellow),
clarity (e.g., clear to slightly opalescent or clear to opalescent), or
viscosity (e.g., between approximately 5 cP and 30 cP when measured at
ambient temperature, such as at 20° C.-30° C.). The post
approval requirement can include a measure of one more of the above
parameters. The method also includes, optionally, determining whether the
observed solution parameter meets a preselected criteria or if the
parameter is in a preselected range; optionally, memorializing the value
or result of the analysis, or communicating with the agency, e.g., by
transmitting the value or result to the regulatory agency.

[0062] In another aspect, the invention features a method of making a
batch of an aqueous formulation of VLA-4 binding antibody having a
preselected property, e.g., meeting a release specification, label
requirement, or compendial requirement, e.g., a property described
herein. The method includes providing a test antibody preparation;
analyzing the test antibody preparation according to a method described
herein; determining if the test antibody preparation satisfies a
preselected criteria, e.g., having a preselected relationship with a
reference value, e.g., one or more reference values disclosed herein, and
selecting the test antibody preparation to make a batch of product.

[0063] In another aspect, the invention features multiple batches of an
aqueous formulation of VLA-4 binding antibody, wherein one or more
solution parameters (e.g., a value or solution parameter determined by a
method described herein), for each batch varies less than a preselected
range from a pre-selected desired reference value or criteria, e.g., a
range or criteria described herein. In some embodiments, one or more
parameters for one or more batches of an antibody formulation, is
determined and a batch or batches selected as a result of the
determination. Some embodiments include comparing the results of the
determination to a preselected value or criteria, e.g., a reference
standard. Other embodiments include adjusting the dose of the batch to be
administered, e.g., based on the result of the determination of the value
or parameter.

[0064] In another aspect, the invention features a method of one or more
of: providing a report to a report-receiving entity, evaluating a sample
of an aqueous formulation of VLA-4 binding antibody for compliance with a
reference standard, e.g., an FDA requirement, seeking indication from
another party that a preparation of the VLA-4 binding antibody meets some
predefined requirement, or submitting information about a preparation of
a VLA-4 binding antibody to another party. Exemplary receiving entities
or other parties include a government, e.g., the U.S. federal government,
e.g., a government agency, e.g., the FDA. The method includes one or more
(or all) of the following steps for making and/or testing an aqueous
formulation of VLA-4 binding antibody in a first country, e.g., the U.S.;
sending at least an aliquot of the sample outside the first country,
e.g., sending it outside the United States, to a second country;
preparing, or receiving, a report which includes data about the structure
of the preparation of the VLA-4 binding antibody, e.g., data related to a
structure and/or chain described herein, e.g., data generated by one or
more of the methods described herein; and providing said report to a
report recipient entity.

[0065] In one embodiment, the report-receiving entity can determine if a
predetermined requirement or reference value is met by the data and,
optionally, a response from the report-receiving entity is received,
e.g., by a manufacturer, distributor or seller of an aqueous formulation
of a VLA-4 binding antibody. In one embodiment, upon receipt of approval
from the report recipient entity, the preparation of VLA-4 binding
antibody is selected, packaged, or placed into commerce.

[0066] In another aspect, the invention features a method of evaluating an
aqueous formulation of VLA-4 binding antibody. The method includes
receiving data with regard to the presence or level of VLA-4 binding
antibody, e.g., wherein the data was prepared by one or more methods
described herein; providing a record which includes said data and
optionally includes an identifier for a batch of VLA-4 binding antibody;
submitting said record to a decision-maker, e.g., a government agency,
e.g., the FDA; optionally, receiving a communication from said decision
maker; optionally, deciding whether to release or market the batch of
VLA-4 binding antibody based on the communication from the decision
maker. In one embodiment, the method further includes releasing the
sample.

[0104] In some embodiments, any of the above formulations 1-7 can be
essentially free of an amino acid, e.g., arginine or glycine, or
glycerol.

[0105] Methods and compositions disclosed herein can be used where the
presence, distribution, or amount, of one or more structures in the
mixture may possess or impinge on the biological activity. The methods
are also useful from a structure-activity prospective, to evaluate or
ensure biological equivalence.

[0107] "Suitable for SC or IM administration" means that administration of
the composition to a subject, such as a human, will have a therapeutic
effect, such as to improve one or more symptoms in the subject.

[0108] The term "treating" refers to administering a therapy in an amount,
manner, and/or mode effective to improve a condition, symptom, or
parameter associated with a disorder or to prevent progression of a
disorder, to either a statistically significant degree or to a degree
detectable to one skilled in the art. An effective amount, manner, or
mode can vary depending on the subject and may be tailored to the
subject.

[0109] A "stable" formulation of VLA-4 binding antibody exhibits little or
no signs of any one or more of aggregation, fragmentation, deamidation,
oxidation, or change in biological activity over an extended period of
time, e.g., 12 months, 24 months, 36 months or longer. For example, in
one embodiment, less than 10% of the composition is aggregated,
fragmented, or oxidated. Aggregation, precipitation, and/or denaturation
can be assessed by known methods, such as visual examination of color
and/or clarity, or by UV light scattering or size exclusion
chromatography. The ability of the protein to retain its biological
activity can be assessed by detecting and quantifying chemically altered
forms of the antibody. Size modification (e.g., clipping), which can be
evaluated using size exclusion chromatography, SDS-PAGE and/or
matrix-assisted laser desorption ionization/time-of-flight mass
spectrometry (MALDI/TOF MS), or peptide mapping of endoproteinase-treated
antibody, for example. Other types of chemical alteration include charge
alteration (e.g., occurring as a result of deamidation), which can be
evaluated by ion-exchange chromatography, for example. An antibody
"retains its biological activity" in a pharmaceutical formulation, if the
biological activity of the antibody at a given time is within about 10%
of the biological activity exhibited at the time the pharmaceutical
formulation was prepared as determined in an antigen binding assay, for
example.

[0110] A "VLA-4 binding antibody" refers to an antibody that binds to a
VLA-4 integrin, such as to the α4 subunit of the VLA-4 integrin,
and at least partially inhibits an activity of VLA-4, particularly a
binding activity of a VLA-4 integrin or a signaling activity, e.g.,
ability to transduce a VLA-4 mediated signal. For example, a VLA-4
binding antibody may inhibit binding of VLA-4 to a cognate ligand of
VLA-4, e.g., a cell surface protein such as VCAM-1, or to an
extracellular matrix component, such as fibronectin or osteopontin. A
VLA-4 binding antibody may bind to either the α4 subunit or the
β1 subunit, or to both. In one embodiment, the antibody binds to the
B1 epitope of α4. A VLA-4 binding antibody may bind to VLA-4 with a
Kd of less than about 10-6, 10-7, 10-8, 10-9, or
10-10 M. VLA-4 is also known as alpha4/beta1 and CD29/CD49b.

[0111] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable region, e.g., an amino acid
sequence that provides an immunoglobulin variable domain or
immunoglobulin variable domain sequence. For example, an antibody can
include a heavy (H) chain variable region (abbreviated herein as VH), and
a light (L) chain variable region (abbreviated herein as VL). In another
example, an antibody includes two heavy

[0112] (H) chain variable regions and two light (L) chain variable
regions. The term "antibody" encompasses antigen-binding fragments of
antibodies (e.g., single chain antibodies, Fab fragments, F(ab')2
fragments, Fd fragments, Fv fragments, and dAb fragments) as well as
complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE,
IgD, IgM (as well as subtypes thereof). The light chains of the
immunoglobulin may be of types kappa or lambda. In one embodiment, the
antibody is glycosylated. An antibody can be functional for antibody
dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be
non-functional for one or both of these activities.

[0113] The VH and VL regions can be further subdivided into regions of
hypervariability, termed "complementarity determining regions" ("CDR"),
interspersed with regions that are more conserved, termed "framework
regions" (FR). The extent of the FRs and CDRs has been precisely defined
(see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services,
NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol.
196:901-917). Kabat definitions are used herein. Each VH and VL is
typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxyl-terminus in the following order: FR1, CDR1,
FR2, CDR2, FR3, CDR3, FR4.

[0115] As used herein, an "immunoglobulin variable domain sequence" refers
to an amino acid sequence that can form the structure of an
immunoglobulin variable domain. For example, the sequence may include all
or part of the amino acid sequence of a naturally-occurring variable
domain. For example, the sequence may omit one, two or more N- or
C-terminal amino acids, internal amino acids, may include one or more
insertions or additional terminal amino acids, or may include other
alterations. In one embodiment, a polypeptide that includes an
immunoglobulin variable domain sequence can associate with another
immunoglobulin variable domain sequence to form a target binding
structure (or "antigen binding site"), e.g., a structure that interacts
with VLA-4.

[0116] The VH or VL chain of the antibody can further include all or part
of a heavy or light chain constant region, to thereby form a heavy or
light immunoglobulin chain, respectively. In one embodiment, the antibody
is a tetramer of two heavy immunoglobulin chains and two light
immunoglobulin chains. The heavy and light immunoglobulin chains can be
connected by disulfide bonds. The heavy chain constant region typically
includes three constant domains, CH1, CH2 and CH3. The light chain
constant region typically includes a CL domain. The variable region of
the heavy and light chains contains a binding domain that interacts with
an antigen. The constant regions of the antibodies typically mediate the
binding of the antibody to host tissues or factors, including various
cells of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.

[0117] One or more regions of an antibody can be human, effectively human,
or humanized. For example, one or more of the variable regions can be
human or effectively human. For example, one or more of the CDRs, e.g.,
HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human
(HC, heavy chain; LC, light chain). Each of the light chain CDRs can be
human. HC CDR3 can be human. One or more of the framework regions can be
human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment,
all the framework regions are human, e.g., derived from a human somatic
cell, e.g., a hematopoietic cell that produces immunoglobulins or a
non-hematopoietic cell. In one embodiment, the human sequences are
germline sequences, e.g., encoded by a germline nucleic acid. One or more
of the constant regions can be human, effectively human, or humanized. In
another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the
framework regions (e.g., FR1, FR2, and FR3, collectively, or FR1, FR2,
FR3, and FR4, collectively) or the entire antibody can be human,
effectively human, or humanized. For example, FR1, FR2, and FR3
collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99%
identical to a human sequence encoded by a human germline segment.

[0118] An "effectively human" immunoglobulin variable region is an
immunoglobulin variable region that includes a sufficient number of human
framework amino acid positions such that the immunoglobulin variable
region does not elicit an immunogenic response in a normal human. An
"effectively human" antibody is an antibody that includes a sufficient
number of human amino acid positions such that the antibody does not
elicit an immunogenic response in a normal human.

[0119] A "humanized" immunoglobulin variable region is an immunoglobulin
variable region that is modified such that the modified form elicits less
of an immune response in a human than does the non-modified form, e.g.,
is modified to include a sufficient number of human framework amino acid
positions such that the immunoglobulin variable region does not elicit an
immunogenic response in a normal human. Descriptions of "humanized"
immunoglobulins include, for example, U.S. Pat. No. 6,407,213 and U.S.
Pat. No. 5,693,762. In some cases, humanized immunoglobulins can include
a non-human amino acid at one or more framework amino acid positions.

[0120] All or part of an antibody can be encoded by an immunoglobulin gene
or a segment thereof. Exemplary human immunoglobulin genes include the
kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4),
delta, epsilon and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Full-length immunoglobulin "light
chains" (about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda
constant region gene at the COOH-terminus. Full-length immunoglobulin
"heavy chains" (about 50 Kd or 446 amino acids), are similarly encoded by
a variable region gene (about 116 amino acids) and one of the other
aforementioned constant region genes, e.g., gamma (encoding about 330
amino acids).

[0121] The term "antigen-binding fragment" of a full length antibody
refers to one or more fragments of a full-length antibody that retain the
ability to specifically bind to a target of interest, e.g., VLA-4.
Examples of binding fragments encompassed within the term
"antigen-binding fragment" of a full length antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1
domains; (ii) a F(ab')2 fragment, a bivalent fragment including two
Fab fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody, (v) a
dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of
a VH domain; and (vi) an isolated complementarity determining region
(CDR) that retains functionality. Furthermore, although the two domains
of the Fv fragment, VL and VH, are coded for by separate genes, they can
be joined, using recombinant methods, by a synthetic linker that enables
them to be made as a single protein chain in which the VL and VH regions
pair to form monovalent molecules known as single chain Fv (scFv). See
e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0122] As used herein, "about" refers to within 0.1% to 5% of the given
value (e.g., within 5%, 3%, 2%, 1%, 0.5%, 0.1% above or below the given
value). Where amounts and other designated values are provided herein,
the allowable deviation is within pharmaceutically acceptable standards.

[0123] Certain advantages are provided by embodiments of the invention. In
some cases, it is difficult to make high concentration formulations of
proteins, e.g., antibodies, for use in pharmaceutical compositions.
Methods of preparing such formulations are presented herein.
Pharmaceutical compositions containing high concentrations of protein,
e.g., of anti-VLA-4 antibody, can be useful for administration over a
shorter time frame. A high concentration formulation, e.g., of anti-VLA-4
antibody, can also be administered by simplified methods (e.g.,
subcutaneously).

[0124] The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent from
the description and drawings, and from the claims.

[0126]FIG. 2 shows the UV absorbance in solution at 340 nm pre- and
post-agitation of natalizumab at a concentration of 150 mg/mL in
formulations as described in FIG. 1.

[0127]FIG. 3 shows the relationship between aggregation and various
levels of polysorbate 80 for natalizumab (150 mg/mL) in the HOL
formulation (HOL=20 mM histidine, 240 mM glycerol, pH 6; polysorbate 80
is a variable of the experiment).

[0128]FIG. 4 shows percentage aggregation over time for natalizumab (150
mg/mL) stored at 40° C. in various formulations as described in
FIG. 1.

[0129]FIG. 5 shows percentage aggregation over time for natalizumab (150
mg/mL) stored in vials between 2-8° C. in various formulations as
described in FIG. 1.

[0130]FIG. 6 shows percentage of methionine oxidation over time for
natalizumab (150 mg/mL) stored between 2-8° C. and at 40°
C. in various formulations as described in FIG. 1.

[0132]FIG. 8 shows fragmentation rates for natalizumab at various
concentrations and in various formulations versus time (8 weeks).

DETAILED DESCRIPTION

[0133] Stable formulations of highly concentrated VLA-4 binding antibody,
are useful for subcutaneous (SC), intramuscular (IM), or intravenous (IV)
administration. The formulations featured in the invention contain from
about 120 mg/mL to about 190 mg/mL VLA-4 binding antibody, such as
natalizumab.

[0134] Pharmaceutical Compositions

[0135] The compositions described herein are formulated as pharmaceutical
compositions. VLA-4 binding antibody (e.g., natalizumab) can be provided,
for example, in a buffered solution at a concentration between about 120
mg/mL and 190 mg/mL (e.g., between about 120 mg/mL and about 180 mg/mL,
between about 140 mg/mL and about 160 mg/mL, between about 135 mg/mL and
about 165 mg/mL; e.g., about 120 mg/mL, 130 mg/mL, 135 mg/mL, 140 mg/mL,
150 mg/mL, 160 mg/mL, 165 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL). In one
embodiment, the VLA-4 binding antibody (e.g., natalizumab) is provided in
a buffered solution at a concentration greater than 150 mg/mL and less
than about 190 mg/mL. In another embodiment, the formulation is prepared
at a higher concentration (e.g., 170 mg/mL to 190 mg/mL), and then
diluted back to the desired concentration (e.g., 135 mg/mL to 165 mg/mL).
For example, the formulation can be prepared with an antibody
concentration of, e.g., 175 mg/mL, 180 mg/mL or 185 mg/mL, and then
diluted back to a concentration desired for administration, e.g., 140
mg/mL, 145 mg/mL, 150 mg/mL, 155 mg/mL, or 160 mg/mL. The composition can
be stored at 2-8° C. (e.g., 4° C., 5° C., 6°
C., 7° C.).

[0136] In one embodiment, the VLA-4 binding antibody can be formulated
with excipient materials, such as 160 mM L-arginine hydrochloride
(±10%), a phosphate buffer (e.g., sodium dibasic phosphate
heptahydrate and sodium monobasic phosphate), and polysorbate 80, where
the total sodium content does not exceed 60 mM. In another embodiment,
VLA-4 binding antibody can be formulated with 275 mM glycerol (±10%),
a phosphate buffer (e.g., sodium dibasic phosphate heptahydrate and
sodium monobasic phosphate or other phosphate salts), and polysorbate 80,
and is substantially free of sodium chloride. In another embodiment,
VLA-4 binding antibody can be formulated with 140 mM sodium chloride
(±10%), a phosphate buffer, and polysorbate 80. Exemplary formulations
that include phosphate buffers are provided below, e.g., at examples 8,
9, 10, 11, and 12.

[0137] In one embodiment, the VLA-4 binding antibody can be formulated
with excipient materials, such as 240 mM glycerol (±10%), a histidine
buffer, polysorbate 80, and optionally L-methionine. Exemplary
formulations that include histidine buffers are provided below, e.g., at
examples 13 and 14.

[0139] Histidine buffers are known in the art and include, e.g., aqueous
solutions of D-histidine, D-histidine monochloride monohydrate,
DL-histidine, DL-histidine monochloride monohydrate, L-histidine, or
L-histidine monochloride monohydrate, brought to the proper pH with
either hydrochloric acid or sodium hydroxide, or other acid or base known
in the art.

[0140] Typically, a pharmaceutical composition includes a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible.

[0141] A "pharmaceutically acceptable salt" refers to a salt that retains
the desired biological activity of the antibody and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J.
Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts
and base addition salts. Acid addition salts include those derived from
nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,
sulfuric, hydrobromic, hydroiodic, and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids, free amino acids, and the
like. Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like, as
well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, procaine and the like.

[0142] Typically physiologically compatible agents, such as free amino
acids, the hydrochloride salts, sodium salts, or potassium salts of free
amino acids are used as excipients in pharmaceutical formulations to
promote stability of the antibody. The formulations herein can include
additives such as glycerol, mannitol, sorbitol, and other polyols, as
well as sugars (e.g., sucrose), to promote stability.

[0143] The formulations featured herein can include a pharmaceutically
acceptable excipient, such as a surfactant, e.g., polysorbate 80,
glycerin monostearate, polyoxyl stearate, lauromacrogol, or sorbitan
oleate. In one embodiment, the formulations featured herein include about
0.01% (w/v) to about 0.1% (w/v) polysorbate 80, e.g., about 0.2% or 0.04%
polysorbate 80.

[0144] The pharmaceutical compositions containing highly concentrated
VLA-4 binding antibodies are in the form of a liquid solution (e.g.,
injectable and infusible solutions). Such compositions can be
administered by a parenteral mode (e.g., subcutaneous, intraperitoneal,
or intramuscular injection). The phrases "parenteral administration" and
"administered parenterally" as used herein mean modes of administration
other than enteral and topical administration, usually by injection, and
include, subcutaneous or intramuscular administration, as well as
intravenous, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcuticular, subcapsular, subarachnoid,
intraspinal, epidural and intrasternal injection and infusion. In one
embodiment, the formulations described herein are administered
subcutaneously.

[0145] Pharmaceutical compositions are sterile and stable under the
conditions of manufacture and storage. A pharmaceutical composition can
also be tested to insure it meets regulatory and industry standards for
administration.

[0146] A pharmaceutical composition containing a highly concentrated
amount of VLA-4 binding antibody can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure suitable
to high antibody concentration. Sterile injectable solutions can be
prepared by incorporating an agent described herein in the required
amount in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating an agent described
herein into a sterile vehicle that contains a basic dispersion medium and
the required other ingredients from those enumerated above. The proper
fluidity of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle
size in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by including
in the composition an agent that delays absorption, for example,
monostearate salts and gelatin.

[0147] In some embodiments, parameters that describe the formulations,
e.g., parameters that may appear on the product label, are characterized.
Such parameters include, e.g., color (typically colorless to slightly
yellow, or colorless to yellow), clarity (typically clear to slightly
opalescent, or clear to opalescent), and viscosity (typically between
about 5 cP and 30 cP when measured at ambient temperature, such as at
20° C. to 30° C.). Such parameters can be measured by
methods known in the art. For example, clarity can be measured using
commercially available opalescence standards (available from, e.g.,
HunterLab Associates, Inc. (Reston, Va.)).

[0150] Formulations containing VLA-4 binding antibody formulations can be
made as described in U.S. Published Application 2005/0053598, modified to
accommodate high concentrations of antibody (e.g., concentrations of
about 75 mg/mL to about 190 mg/mL, 100 mg/mL to about 180 mg/mL, about
120 mg/mL to about 170 mg/mL, 135 mg/mL to about 165 mg/mL). The process
can be altered as would be known to the skilled artisan, but generally
would follow a procedure such as the following. Obtain an ampoule from a
working cell bank containing cells that make the antibody or protein of
interest. Prepare an inoculum. Culture or ferment the cells of the
inoculum with additional feedings as is necessary. Harvest/clarify the
cells by centrifugation and/or filtration. This can be done for example
by concentrating the cells 10 fold by, e.g., spiral wound filtration.
Intermediate filtration, such as through a 0.2 μm filter, is followed
by, e.g., affinity chromatography, such as by a protein A Sepharose Fast
Flow®, and then reverse elution. The antibody containing composition
then receives a treatment at low pH, such as at pH 3.6-3.7. The mixture
then receives a viral filtration followed by a
concentration/diafiltration step. The composition is further purified by,
e.g., anion exchange chromatography, such as by DEAE Sepharose Fast
Flow®. This step can be performed multiple times. From this point,
the composition is then further concentrated and then purified, e.g., by
gel filtration chromatrography, such as through a Sephacryl S300HR®
system. The antibody containing composition can be further concentrated
if so desired. The final formulation is produced by adding buffer and
polysorbate, and concentrating the antibody again through an
ultrafiltration process. The resulting antibody formulation can be
quality control tested and quality assurance (QA) released. An antibody
formulation can be produced according to any of the methods exemplified
in Table 1 below. For example, the formulation containing a VLA-4 binding
antibody, such as natalizumab, can be produced by the following process.
A large batch of cell culture, such as from 5,000 to 20,000 Liters (e.g.,
5,000; 10,000; 15,000; 20,000 Liters) is inoculated, cultured, fed,
harvested and clarified as known in the art. The clarified material is
purified e.g., by chromatography, viral inactivation, and viral
filtration. Ultrafiltation/diafiltration (UF/DF) of the clarified
material results in a phosphate process intermediate. The phosphate
process intermediate may be stored at 2-8° C., e.g., for future
processing, such as by methods to further concentrate the protein. To
make the final formulation, polysorbate and buffer (as described herein)
are added to the phosphate process intermediate to achieve the final
desired antibody concentration. In one alternative, the final formulation
is created by backdiluting the phosphate process intermediate into buffer
to a final desired concentration, e.g., a low concentration such as 20
mg/mL. Polysorbate is typically added during the final dilution step.

[0151] In one embodiment, the VLA-4 binding antibody formulation is
produced in a histidine formulation, as described above, except that the
phosphate process intermediate undergoes at least a second UF/DF process,
and optionally, at least one additional UF process, into a histidine
forumulation buffer as described herein, to a final desired
concentration, such as between about 75 mg/mL and 190 mg/mL, e.g., about
75 mg/mL to about 190 mg/mL, e.g., 75 mg/mL, 100 mg/mL, 125 mg/mL, 135
mg/mL, 150 mg/mL, 165 mg/mL, 180 mg/mL, 190 mg/mL. In one alternative,
the antibody formulation in histidine buffer is brought to a final
concentration greater than a desired concentration. Then the final
formulation is created by backdiluting into histidine formulation buffer
to a desired protein concentration. Polysorbate is typically added during
the final dilution step.

[0152] In another embodiment, the antibody formulation is produced in a
phosphate formulation, as described above, except that the phosphate
process intermediate undergoes at least a second UF/DF process, and
optionally, at least a one additional UF process, into a phosphate
formulation buffer as described herein, to a final desired concentration,
such as between about 75 mg/mL and 190 mg/mL, e.g., about 75 mg/mL to
about 190 mg/mL, e.g., 75 mg/mL, 100 mg/mL, 125 mg/mL, 135 mg/mL, 150
mg/mL, 165 mg/mL, 180 mg/mL, 190 mg/mL. In one alternative, the antibody
formulation in phosphate buffer is brought to a final concentration
greater than the desired concentration, and the final formulation is
created by backdiluting into phosphate buffer to the desired
concentration. Polysorbate is typically added during the final dilution
step.

[0153] In another embodiment, the VLA-4 binding antibody formulation is
produced in a phosphate formulation, as described above, except that a
commercial UF/DF process for the phosphate formulation is followed, and
polysorbate added, to produce a VLA-4 binding antibody formulation at a
desired final concentration, such as between about 75 mg/mL and 190
mg/mL, e.g., about 75 mg/mL to about 190 mg/mL, e.g., 75 mg/mL, 100
mg/mL, 125 mg/mL, 135 mg/mL, 150 mg/mL, 165 mg/mL, 180 mg/mL, 190 mg/mL.
In one alternative, the antibody formulation in phosphate buffer is
brought to a final concentration greater than the desired concentration,
and then the final formulation is created by backdiluting into phosphate
formulation buffer to the desired protein concentration. Polysorbate is
typically added during the final dilution process.

[0155] The commercial UF/DF process of Table 1 typically uses a first
UF/DF operation to diafilter the product into a formulation buffer and
concentrate the product to about 5 g/L to 40 g/L (e.g., about 10 g/L,
about 20 g/L, about 30 g/L). A second UF operation is typically performed
to further concentrate the product to about 135 g/L to about 185 g/L
(e.g., about 140 g/L, 150 g/L, 165 g/L).

[0156] Any of the VLA-4 binding antibody formulations described herein can
be packaged in aseptic vials as described in, e.g., U.S. Published
Application 2005/0053598, which is incorporated herein by reference. For
example, the formulations can be packaged in, e.g., 3.0, 5.0 or 20 mL
fill vials. Filled drug product is stored under refrigeration at about
2-8° C.

[0157] In one embodiment, the final formulation is packaged as a liquid in
a 3.0 mL fill vial with an extractable minimum volume of 1 mL. For
example, the fill vial can include about 1.1 mL to about 1.5 mL (e.g.,
about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL) of antibody
formulation. In another embodiment, that antibody formulation is packaged
in a pre-filled syringe, in an amount such that 1 mL of solution is
injected into a patient upon use, and the 1 mL solution delivers the
desired amount of antibody, e.g., 135 mg to 165 mg natalizumab, e.g., 150
mg natalizumab.

[0158] Natalizumab and Other VLA-4 Binding Antibodies

[0159] Antibodies suitable for a highly concentrated VLA-4 binding
antibody formulation described herein include natalizumab, an α4
integrin binding antibody. Natalizumab (USAN name) has the antibody code
number AN100226, and is also called "TYSABRI®" The amino acid sequence
of the light chain and heavy chain of natalizumab prior to any in vivo
modifications (e.g., clipping of amino acids) is shown in Table 1-1 and
Table 1-2.

[0160] Natalizumab inhibits the migration of leukocytes from the blood to
the central nervous system. Natalizumab binds to VLA-4 (also called
α4β1) on the surface of activated T-cells and other
mononuclear leukocytes. It can disrupt adhesion between the T-cell and
endothelial cells, and thus prevent migration of mononuclear leukocytes
across the endothelium and into the parenchyma. As a result, the levels
of proinflammatory cytokines can also be reduced.

[0161] Natalizumab can decrease the number of brain lesions and clinical
relapses in patients with relapse remitting multiple sclerosis and
relapsing secondary-progressive multiple sclerosis.

[0163] Some VLA-4 binding antibodies recognize epitopes of the a4 subunit
that are involved in binding to a cognate ligand, e.g., VCAM-1 or
fibronectin. Many such antibodies inhibit binding of VLA-4 to cognate
ligands (e.g., VCAM-1 and fibronectin).

[0164] Some useful VLA-4 binding antibodies can interact with VLA-4 on
cells, e.g., lymphocytes, but do not cause cell aggregation. However,
other VLA-4 binding antibodies have been observed to cause such
aggregation. HP1/2 does not cause cell aggregation. The HP1/2 monoclonal
antibody (Sanchez-Madrid et al., 1986) has an extremely high potency,
blocks VLA-4 interaction with both VCAM1 and fibronectin, and has the
specificity for epitope B on VLA-4. This antibody and other B
epitope-specific antibodies (such as B1 or B2 epitope binding antibodies;
Pulido et al., 1991, supra) represent one class of VLA-4 binding
antibodies that can be used in the formulations and methods described
herein.

[0165] An exemplary VLA-4 binding antibody has one or more CDRs, e.g., all
three HC CDRs and/or all three LC CDRs of a particular antibody disclosed
herein, or CDRs that are, in sum, at least 80, 85, 90, 92, 94, 95, 96,
97, 98, 99% identical to such an antibody, e.g., natalizumab. In one
embodiment, the H1 and H2 hypervariable loops have the same canonical
structure as those of an antibody described herein. In one embodiment,
the L1 and L2 hypervariable loops have the same canonical structure as
those of an antibody described herein.

[0166] In one embodiment, the amino acid sequence of the HC and/or LC
variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97, 98, 99,
or 100% identical to the amino acid sequence of the HC and/or LC variable
domain of an antibody described herein, e.g., natalizumab. The amino acid
sequence of the HC and/or LC variable domain sequence can differ by at
least one amino acid, but no more than ten, eight, six, five, four,
three, or two amino acids from the corresponding sequence of an antibody
described herein, e.g., natalizumab. For example, the differences may be
primarily or entirely in the framework regions.

[0167] The amino acid sequences of the HC and LC variable domain sequences
can be encoded by a nucleic acid sequence that hybridizes under high
stringency conditions to a nucleic acid sequence described herein or one
that encodes a variable domain or an amino acid sequence described
herein. In one embodiment, the amino acid sequences of one or more
framework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC and/or LC
variable domain are at least 70, 80, 85, 90, 92, 95, 97, 98, 99, or 100%
identical to corresponding framework regions of the HC and LC variable
domains of an antibody described herein. In one embodiment, one or more
heavy or light chain framework regions (e.g., HC FR1, FR2, and FR3) are
at least 70, 80, 85, 90, 95, 96, 97, 98, or 100% identical to the
sequence of corresponding framework regions from a human germline
antibody.

[0168] Calculations of "homology" or "sequence identity" between two
sequences (the terms are used interchangeably herein) are performed as
follows. The sequences are aligned for optimal comparison purposes (e.g.,
gaps can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). The optimal
alignment is determined as the best score using the GAP program in the
GCG software package with a Blossum 62 scoring matrix with a gap penalty
of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The
amino acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the first
sequence is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules are
identical at that position (as used herein amino acid or nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology"). The
percent identity between the two sequences is a function of the number of
identical positions shared by the sequences.

[0169] As used herein, the term "hybridizes under high stringency
conditions" describes conditions for hybridization and washing. Guidance
for performing hybridization reactions can be found in Current Protocols
in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which
is incorporated by reference. Aqueous and nonaqueous methods are
described in that reference and either can be used. High stringency
hybridization conditions include hybridization in 6×SSC at about
45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS
at 65° C., or substantially similar conditions.

[0170] Administration

[0171] The highly concentrated VLA-4 binding antibody formulations
described herein can be administered to a subject, e.g., a human subject,
by a variety of methods, including subcutaneous, intramuscular and
intravenous administration. Typically, administration is by subcutaneous
or intramuscular injection.

[0172] The formulation can be administered as a fixed dose, or in a mg/kg
dose. Typically the administration is in a fixed dose. For example, the
formulation is administered at a fixed unit dose of between 75 mg and 500
mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg)
every 4 weeks (e.g., monthly), or between 50 mg and 250 mg (e.g., 75 mg,
100 mg, 150 mg, 200 mg) every two weeks, or between 25 mg and 150 mg
(e.g., 50 mg, 75 mg, 100 mg, 125 mg) once a week. The formulation can
also be administered in a bolus at a dose of between 1 and 8 mg/kg, e.g.,
about 6.0, 4.0, 3.0, 2.0, 1.0 mg/kg. Modified dose ranges include a dose
that is less than 500, 400, 300, 250, 200, 150 or 100 mg/subject,
typically for administration every fourth week or once a month. The VLA-4
binding antibody can be administered, for example, every three to five
weeks, e.g., every fourth week, or monthly.

[0173] Dosage regimens can be adjusted to provide the desired response,
e.g., a therapeutic response. The dose can also be chosen to reduce or
avoid production of antibodies against the VLA-4 binding antibody, to
achieve greater than 40, 50, 70, 75, or 80% saturation of the α4
subunit, to achieve to less than 80%, 70%, 60%, 50%, or 40% saturation of
the α4 subunit, or to prevent an increase the level of circulating
white blood cells.

[0174] In certain embodiments, the active agent can be prepared with a
carrier that will protect the antibody against rapid release, such as a
controlled release formulation, including implants, and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Many methods for the preparation of
such formulations are patented or generally known. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel
Dekker, Inc., New York, 1978.

[0175] Dosage regimens can be adjusted to provide the desired response,
e.g., a therapeutic response. A "therapeutic response" is an improvement
in a condition, symptom, or parameter associated with a disorder, to
either a statistically significant degree or to a degree detectable to
one skilled in the art.

[0176] Dosage unit form or "fixed dose" as used herein refers to
physically discrete units suited as unitary dosages for the subjects to
be treated; each unit contains a predetermined quantity of active
antibody calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier and optionally in
association with the other agent.

[0177] A pharmaceutical composition may include a "therapeutically
effective amount" of a VLA-4-binding antibody, e.g. natalizumab,
described herein. Such effective amounts can be determined based on the
effect of the administered agent, or the combinatorial effect of an agent
and secondary agent if more than one agent is used. A therapeutically
effective amount of an agent may also vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of the antibody to elicit a desired response in the individual,
e.g., amelioration of at least one disorder parameter, e.g., a multiple
sclerosis parameter, or amelioration of at least one symptom of the
disorder, e.g., multiple sclerosis. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the composition are
outweighed by the therapeutically beneficial effects.

[0178] Devices and Kits

[0179] Formulations having a high concentration of a VLA-4-binding
antibody (e.g., natalizumab) can be administered with a medical device.
The device can be designed with or have features such as portability,
room temperature storage, and ease of use so that it can be used in
emergency situations, e.g., by an untrained subject or by emergency
personnel in the field, removed to medical facilities and other medical
equipment. The device can include, e.g., one or more housings for storing
pharmaceutical preparations that include a VLA-4-binding antibody (e.g.,
natalizumab), and can be configured to deliver one or more unit doses of
the agent.

[0180] For example, the pharmaceutical composition can be administered
with a transcutaneous delivery device, such as a syringe, including a
hypodermic or multichamber syringe. In one embodiment, the device is a
prefilled syringe with attached or integral needle. In other embodiments,
the device is a prefilled syringe not having a needle attached. The
needle can be packaged with the prefilled syringe. In one embodiment, the
device is an auto-injection device, e.g., an auto-injector syringe. In
another embodiment the injection device is a pen-injector. In yet another
embodiment, the syringe is a staked needle syringe, luer lock syringe, or
luer slip syringe. Other suitable delivery devices include stents,
catheters, microneedles, and implantable controlled release devices. The
composition can be administered intravenously with standard IV equipment,
including, e.g., IV tubings, with or without in-line filters. In certain
embodiments, the device will be a syringe for use in SC or IM
administration.

[0181] Pharmaceutical compositions can be administered with medical
devices. For example, pharmaceutical compositions can be administered
with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413,
4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and
modules include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate; U.S.
Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat. No.
4,475,196, which discloses an osmotic drug delivery system. The
therapeutic composition can also be in the form of a biodegradable or
nonbiodegradable sustained release formulation for subcutaneous or
intramuscular administration. See, e.g., U.S. Pat. Nos. 3,773,919 and
4,767,628 and PCT Application No. WO 94/15587. Continuous administration
can also be achieved using an implantable or external pump. The
administration can also be conducted intermittently, e.g., single daily
injection, or continuously at a low dose, e.g., sustained release
formulation. The delivery device can be modified to be optimally suited
for administration of VLA-4 binding antibody. For example, a syringe can
be siliconized to an extent that is optimal for storage and delivery of
anti-VLA-4 antibody. Of course, many other such implants, delivery
systems, and modules are also known.

[0182] The invention also features a device for administering a first and
second agent. The device can include, e.g., one or more housings for
storing pharmaceutical preparations, and can be configured to deliver
unit doses of the first and second agent. The first and second agents can
be stored in the same or separate compartments. For example, the device
can combine the agents prior to administration. It is also possible to
use different devices to administer the first and second agent.

[0183] A VLA-4-binding antibody (e.g., natalizumab) can be provided in a
kit. In one embodiment, the kit includes (a) a container that contains a
composition that includes a high concentration of VLA-4-binding antibody,
optionally (b) a container that contains a composition that includes a
second agent and optionally (c) informational material. The informational
material can be descriptive, instructional, marketing or other material
that relates to the methods described herein and/or the use of the agents
for therapeutic benefit. In one embodiment, the kit also includes a
second agent. For example, the kit includes a first container that
contains a composition that includes the VLA-4-binding antibody, and a
second container that includes the second agent. In one embodiment, the
kit includes one or more single-use syringes pre-filled with a high
concentration liquid antibody formulation described herein.

[0184] The informational material of the kits is not limited in its form.
In one embodiment, the informational material can include information
about production of the antibody, concentration, date of expiration,
batch or production site information, and so forth. In one embodiment,
the informational material relates to methods of administering the
VLA-4-binding antibody (e.g., natalizumab), e.g., in a suitable dose,
dosage form, or mode of administration (e.g., a dose, dosage form, or
mode of administration described herein), to treat a subject who has an
inflammatory disease (e.g., MS), or who is at risk for experiencing an
episode associated with an inflammatory disease. The information can be
provided in a variety of formats, including printed text, computer
readable material, video recording, or audio recording, or information
that provides a link or address to substantive material.

[0185] In addition to the agent, the composition in the kit can include
other ingredients, such as a solvent or buffer, a stabilizer, or a
preservative. The agent can be provided in any form, e.g., liquid, dried
or lyophilized form, and in substantially pure and/or sterile form. When
the agents are provided in a liquid solution, the liquid solution is, for
example, an aqueous solution. When the agents are provided as a dried
form, reconstitution generally is by the addition of a suitable solvent.
The solvent, e.g., sterile water or buffer, can optionally be provided in
the kit.

[0186] The kit can include one or more containers for the composition or
compositions containing the agents. In some embodiments, the kit contains
separate containers, dividers or compartments for the composition and
informational material. For example, the composition can be contained in
a bottle, vial, or syringe, and the informational material can be
contained in a plastic sleeve or packet. In other embodiments, the
separate elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle, vial or
syringe that has attached thereto the informational material in the form
of a label. In some embodiments, the kit includes a plurality (e.g., a
pack) of individual containers, each containing one or more unit dosage
forms (e.g., a dosage form described herein) of the agents. The
containers can include a combination unit dosage, e.g., a unit that
includes both the VLA-4-binding antibody (e.g., natalizumab) and the
second agent, e.g., in a desired ratio. For example, the kit includes a
plurality of syringes, ampoules, foil packets, blister packs, or medical
devices, e.g., each containing a single combination unit dose. The
containers of the kits can be air tight, waterproof (e.g., impermeable to
changes in moisture or evaporation), and/or light-tight.

[0187] The kit optionally includes a device suitable for administration of
the composition, e.g., a syringe or other suitable delivery device. The
device can be provided pre-loaded with one or both of the agents or can
be empty, but suitable for loading.

[0188] Multiple Sclerosis

[0189] Formulations having highly concentrated VLA-4 binding antibody
suitable for SC or IM administration are useful for the treatment of
inflammatory diseases, such as multiple sclerosis (MS). Multiple
sclerosis is a central nervous system disease that is characterized by
inflammation and loss of myelin sheaths.

[0190] Patients having MS may be identified by criteria establishing a
diagnosis of clinically definite MS as defined by the workshop on the
diagnosis of MS (Poser et al., Ann. Neurol. 13:227, 1983). Briefly, an
individual with clinically definite MS has had two attacks and clinical
evidence of either two lesions or clinical evidence of one lesion and
paraclinical evidence of another, separate lesion. Definite MS may also
be diagnosed by evidence of two attacks and oligoclonal bands of IgG in
cerebrospinal fluid or by combination of an attack, clinical evidence of
two lesions and oligoclonal band of IgG in cerebrospinal fluid. The
McDonald criteria can also be used to diagnose MS. (McDonald et al.,
2001, Recommended diagnostic criteria for multiple sclerosis: guidelines
from the International Panel on the Diagnosis of Multiple Sclerosis, Ann
Neurol 50:121-127). The McDonald criteria include the use of MRI evidence
of CNS impairment over time to be used in diagnosis of MS, in the absence
of multiple clinical attacks. Effective treatment of multiple sclerosis
may be evaluated in several different ways. The following parameters can
be used to gauge effectiveness of treatment. Two exemplary criteria
include: EDSS (extended disability status scale), and appearance of
exacerbations on MRI (magnetic resonance imaging). The EDSS is a means to
grade clinical impairment due to MS (Kurtzke, Neurology 33:1444, 1983).
Eight functional systems are evaluated for the type and severity of
neurologic impairment. Briefly, prior to treatment, patients are
evaluated for impairment in the following systems: pyramidal, cerebella,
brainstem, sensory, bowel and bladder, visual, cerebral, and other.
Follow-ups are conducted at defined intervals. The scale ranges from 0
(normal) to 10 (death due to MS). A decrease of one full step indicates
an effective treatment (Kurtzke, Ann. Neurol. 36:573-79, 1994).

[0191] Exacerbations are defined as the appearance of a new symptom that
is attributable to MS and accompanied by an appropriate new neurologic
abnormality (IFNB MS Study Group, supra). In addition, the exacerbation
must last at least 24 hours and be preceded by stability or improvement
for at least 30 days. Briefly, patients are given a standard neurological
examination by clinicians. Exacerbations are either mild, moderate, or
severe according to changes in a Neurological Rating Scale (Sipe et al.,
Neurology 34:1368, 1984). An annual exacerbation rate and proportion of
exacerbation-free patients are determined.

[0192] Therapy can be deemed to be effective if there is a statistically
significant difference in the rate or proportion of exacerbation-free or
relapse-free patients between the treated group and the placebo group for
either of these measurements. In addition, time to first exacerbation and
exacerbation duration and severity may also be measured. A measure of
effectiveness as therapy in this regard is a statistically significant
difference in the time to first exacerbation or duration and severity in
the treated group compared to control group. An exacerbation-free or
relapse-free period of greater than one year, 18 months, or 20 months is
particularly noteworthy.

[0193] Efficacy of administering a first agent and, optionally, a second
agent, can also be evaluated based on one or more of the following
criteria: frequency of MBP reactive T cells determined by limiting
dilution, proliferation response of MBP reactive T cell lines and clones,
cytokine profiles of T cell lines and clones to MBP established from
patients. Efficacy is indicated by decrease in frequency of reactive
cells, a reduction in thymidine incorporation with altered peptide
compared to native, and a reduction in TNF and IFN-α.

[0194] Clinical measurements include the relapse rate in one and two-year
intervals, and a change in EDSS, including time to progression from
baseline of 1.0 unit on the EDSS that persists for six months. On a
Kaplan-Meier curve, a delay in sustained progression of disability shows
efficacy. Other criteria include a change in area and volume of T2 images
on MRI, and the number and volume of lesions determined by gadolinium
enhanced images.

[0195] MRI can be used to measure active lesions using
gadolinium-DTPA-enhanced imaging (McDonald et al. Ann. Neurol. 36:14,
1994) or the location and extent of lesions using T2--weighted
techniques. Briefly, baseline MRIs are obtained. The same imaging plane
and patient position are used for each subsequent study. Positioning and
imaging sequences can be chosen to maximize lesion detection and
facilitate lesion tracing. The same positioning and imaging sequences can
be used on subsequent studies. The presence, location and extent of MS
lesions can be determined by radiologists. Areas of lesions can be
outlined and summed slice by slice for total lesion area. Three analyses
may be done: evidence of new lesions, rate of appearance of active
lesions, percentage change in lesion area (Paty et al., Neurology 43:665,
1993). Improvement due to therapy can be established by a statistically
significant improvement in an individual patient compared to baseline or
in a treated group versus a placebo group.

[0197] Each case of MS displays one of several patterns of presentation
and subsequent course. Most commonly, MS first manifests itself as a
series of attacks followed by complete or partial remissions as symptoms
mysteriously lessen, only to return later after a period of stability.
This is called relapsing-remitting (RR) MS. Primary-progressive (PP) MS
is characterized by a gradual clinical decline with no distinct
remissions, although there may be temporary plateaus or minor relief from
symptoms. Secondary-progressive (SP) MS begins with a relapsing-remitting
course followed by a later primary-progressive course. Rarely, patients
may have a progressive-relapsing (PR) course in which the disease takes a
progressive path punctuated by acute attacks. PP, SP, and PR are
sometimes lumped together and called chronic progressive MS.

[0198] A few patients experience malignant MS, defined as a swift and
relentless decline resulting in significant disability or even death
shortly after disease onset. This decline may be arrested or decelerated
by administration of a combination therapy described herein.

[0199] In addition to or prior to human studies, an animal model can be
used to evaluate the efficacy of using the two agents. An exemplary
animal model for multiple sclerosis is the experimental autoimmune
encephalitis (EAE) mouse model, e.g., as described in (Tuohy et al. (J.
Immunol. (1988) 141: 1126-1130), Sobel et al. (J. Immunol. (1984) 132:
2393-2401), and Traugott (Cell Immunol. (1989) 119: 114-129). Mice can be
administered a first and second agent described herein prior to EAE
induction. Then the mice are evaluated for characteristic criteria to
determine the efficacy of using the two agents in the model.

[0202] For example, a formulation containing a high concentration of VLA-4
binding antibody, (e.g., natalizumab) can be administered subcutaneously
or intramuscularly to treat these and other inflammatory, immune, or
autoimmune disorders.

[0203] Antibody Generation

[0204] Antibodies that bind to VLA-4 can be generated by immunization,
e.g., using an animal, or by in vitro methods such as phage display. All
or part of VLA-4 can be used as an immunogen. For example, the
extracellular region of the α4 subunit can be used as an immunogen.
In one embodiment, the immunized animal contains immunoglobulin producing
cells with natural, human, or partially human immunoglobulin loci. In one
embodiment, the non-human animal includes at least a part of a human
immunoglobulin gene. For example, it is possible to engineer mouse
strains deficient in mouse antibody production with large fragments of
the human Ig loci. Using the hybridoma technology, antigen-specific
monoclonal antibodies derived from the genes with the desired specificity
may be produced and selected. See, e.g., XenoMouse®, Green et al.
Nature Genetics 7:13-21 (1994), U.S. 2003-0070185, U.S. Pat. No.
5,789,650, and WO 96/34096.

[0205] Non-human antibodies to VLA-4 can also be produced, e.g., in a
rodent. The non-human antibody can be humanized, e.g., as described in
U.S. Pat. No. 6,602,503, EP 239 400, U.S. Pat. No. 5,693,761, and U.S.
Pat. No. 6,407,213.

[0206] EP 239 400 (Winter et al.) describes altering antibodies by
substitution (within a given variable region) of their complementarity
determining regions (CDRs) for one species with those from another.
CDR-substituted antibodies can be less likely to elicit an immune
response in humans compared to true chimeric antibodies because the
CDR-substituted antibodies contain considerably less non-human
components. (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et
al., 1988, Science 239, 1534-1536). Typically, CDRs of a murine antibody
substituted into the corresponding regions in a human antibody by using
recombinant nucleic acid technology to produce sequences encoding the
desired substituted antibody. Human constant region gene segments of the
desired isotype (usually gamma I for CH and kappa for CL) can be added
and the humanized heavy and light chain genes can be co-expressed in
mammalian cells to produce soluble humanized antibody.

[0207] Queen et al., 1989 and WO 90/07861 have described a process that
includes choosing human V framework regions by computer analysis for
optimal protein sequence homology to the V region framework of the
original murine antibody, and modeling the tertiary structure of the
murine V region to visualize framework amino acid residues that are
likely to interact with the murine CDRs. These murine amino acid residues
are then superimposed on the homologous human framework. See also U.S.
Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101. Tempest et al.,
1991, Biotechnology 9, 266-271, utilize, as standard, the V region
frameworks derived from NEWM and REI heavy and light chains,
respectively, for CDR-grafting without radical introduction of mouse
residues. An advantage of using the Tempest et al. approach to construct
NEWM and REI based humanized antibodies is that the three dimensional
structures of NEWM and REI variable regions are known from X-ray
crystallography and thus specific interactions between CDRs and V region
framework residues can be modeled.

[0209] Fully human monoclonal antibodies that bind to VLA-4 can be
produced, e.g., using in vitro-primed human splenocytes, as described by
Boerner et al., 1991, J. Immunol., 147, 86-95. They may be prepared by
repertoire cloning as described by Persson et al., 1991, Proc. Nat. Acad.
Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol.
Methods 141, 227-236; also U.S. Pat. No. 5,798,230. Large nonimmunized
human phage display libraries may also be used to isolate high affinity
antibodies that can be developed as human therapeutics using standard
phage technology (see, e.g., Vaughan et al, 1996; Hoogenboom et al.
(1998) Immunotechnology 4:1-20; and Hoogenboom et al. (2000) Immunol
Today 2:371-8; U.S. 2003-0232333).

[0210] Antibody Production

[0211] Antibodies can be produced in prokaryotic and eukaryotic cells. In
one embodiment, the antibodies (e.g., scFvs) are expressed in a yeast
cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol Methods.
251:123-35), Hanseula, or Saccharomyces.

[0213] In addition to the nucleic acid sequence encoding the
immunoglobulin domain, the recombinant expression vectors may carry
additional nucleic acid sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of replication)
and selectable marker genes. The selectable marker gene facilitates
selection of host cells into which the vector has been introduced (see
e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). Exemplary
selectable marker genes include the dihydrofolate reductase (DHFR) gene
(for use in dhfr.sup.- host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).

[0214] In an exemplary system for recombinant expression of an antibody
(e.g., a full length antibody or an antigen-binding portion thereof), a
recombinant expression vector encoding both the antibody heavy chain and
the antibody light chain is introduced into dhfr- CHO cells by calcium
phosphate-mediated transfection. Within the recombinant expression
vector, the antibody heavy and light chain genes are each operatively
linked to enhancer/promoter regulatory elements (e.g., derived from SV40,
CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter
regulatory element or an SV40 enhancer/AdMLP promoter regulatory element)
to drive high levels of transcription of the genes. The recombinant
expression vector also carries a DHFR gene, which allows for selection of
CHO cells that have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light chains
and intact antibody is recovered from the culture medium. Standard
molecular biology techniques are used to prepare the recombinant
expression vector, to transfect the host cells, to select for
transformants, to culture the host cells, and to recover the antibody
from the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G. For example,
purified VLA-4-binding antibodies, e.g. natalizumab, can be concentrated
to about 100 mg/mL to about 200 mg/mL using standard protein
concentration techniques.

[0215] Antibodies may also include modifications, e.g., modifications that
alter Fc function, e.g., to decrease or remove interaction with an Fc
receptor or with Clq, or both. For example, the human IgG1 constant
region can be mutated at one or more residues, e.g., one or more of
residues 234 and 237, e.g., according to the numbering in U.S. Pat. No.
5,648,260. Other exemplary modifications include those described in U.S.
Pat. No. 5,648,260.

[0216] For some antibodies that include an Fc domain, the antibody
production system may be designed to synthesize antibodies in which the
Fc region is glycosylated. For example, the Fc domain of IgG molecules is
glycosylated at asparagine 297 in the CH2 domain. This asparagine is the
site for modification with biantennary-type oligosaccharides. This
glycosylation participates in effector functions mediated by Fcγ
receptors and complement Clq (Burton and Woof (1992) Adv. Immunol.
51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). The Fc domain
can be produced in a mammalian expression system that appropriately
glycosylates the residue corresponding to asparagine 297. The Fc domain
can also include other eukaryotic post-translational modifications.

[0217] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method for expressing an
antibody in the mammary gland of a transgenic mammal. A transgene is
constructed that includes a milk-specific promoter and nucleic acid
sequences encoding the antibody of interest, e.g., an antibody described
herein, and a signal sequence for secretion. The milk produced by females
of such transgenic mammals includes, secreted-therein, the antibody of
interest, e.g., an antibody described herein. The antibody can be
purified from the milk, or for some applications, used directly.

[0218] Antibodies can be modified, e.g., with a moiety that improves its
stabilization and/or retention in circulation, e.g., in blood, serum,
lymph, bronchoalveolar lavage, or other tissues, e.g., by at least 1.5,
2, 5, 10, or 50 fold.

[0219] For example, a VLA-4 binding antibody can be associated with a
polymer, e.g., a substantially non-antigenic polymer, such as a
polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary
substantially by weight. Polymers having molecular number average weights
ranging from about 200 to about 35,000 daltons (or about 1,000 to about
15,000, and 2,000 to about 12,500) can be used.

[0220] For example, a VLA-4 binding antibody can be conjugated to a water
soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.
polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of such
polymers include polyalkylene oxide homopolymers such as polyethylene
glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols,
copolymers thereof and block copolymers thereof, provided that the water
solubility of the block copolymers is maintained. Additional useful
polymers include polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block copolymers of polyoxyethylene and
polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or
unbranched polysaccharides that comprise the saccharide monomers
D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid
(e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,
dextran, dextrins, glycogen, or the polysaccharide subunit of acid
mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols
such as polysorbitol and polymannitol; heparin or heparon.

[0221] Exemplary Second Agents

[0222] In some cases, the formulations described herein, e.g.,
formulations containing a high concentration of VLA-4 binding antibody
suitable for SC or IM administration, include a second agent, or are
administered in combination with a formulation containing a second agent.

[0223] In one implementation, the VLA-4 binding antibody and second agent
is provided as a co-formulation, and the co-formulation is administered
to the subject. It is further possible, e.g., at least 24 hours before or
after administering the co-formulation, to administer separately one dose
of the highly concentrated antibody formulation and then one dose of a
formulation containing the second agent. In another implementation, the
antibody and the second agent are provided as separate formulations, and
the step of administering includes sequentially administering the
antibody and the second agent. The sequential administrations can be
provided on the same day (e.g., within one hour of one another or at
least 3, 6, or 12 hours apart) or on different days.

[0224] Generally, the antibody and the second agent are each administered
as a plurality of doses separated in time. The antibody and the second
agent are generally each administered according to a regimen. The regimen
for one or both may have a regular periodicity. The regimen for the
antibody can have a different periodicity from the regimen for the second
agent, e.g., one can be administered more frequently than the other. In
one implementation, one of the antibody and the second agent is
administered once weekly and the other once monthly. In another
implementation, one of the antibody and the second agent is administered
continuously, e.g., over a period of more than 30 minutes but less than
1, 2, 4, or 12 hours, and the other is administered as a bolus. The
antibody and the second agent can be administered by any appropriate
method, e.g., subcutaneously, intramuscularly, or intravenously.

[0225] In some embodiments, each of the antibody and the second agent is
administered at the same dose as each is prescribed for monotherapy. In
other embodiments, the antibody is administered at a dosage that is equal
to or less than an amount required for efficacy if administered alone
Likewise, the second agent can be administered at a dosage that is equal
to or less than an amount required for efficacy if administered alone.

[0236] Glatiramer acetate is a protein formed of a random chain of amino
acids--glutamic acid, lysine, alanine and tyrosine (hence GLATiramer). It
can be synthesized in solution from these amino acids a ratio of
approximately 5 parts alanine to 3 of lysine, 1.5 of glutamic acid and 1
of tyrosine using N-carboxyamino acid anhydrides.

[0238] Still other exemplary antibodies include antibodies that provide an
activity of an agent described herein, e.g., an antibody that engages an
interferon receptor, e.g., an interferon beta receptor. Typically, in
implementations in which the second agent includes an antibody, it binds
to a target protein other tha VLA-4 or other than α4 integrin, or
at least an epitope on VLA-4 other than one recognized by the first
agent.

[0242] In addition to a second agent, it is also possible to deliver still
other agents to the subject. However, in some embodiments, no protein or
no biologic, other than the VLA-4 binding antibody and second agent, are
administered to the subject as a pharmaceutical composition. The VLA-4
binding antibody and the second agent may be the only agents that are
delivered by injection. In embodiments in which the VLA-4 binding
antibody and the second agent are recombinant proteins, the VLA-4 binding
antibody and second agent may be the only recombinant agents administered
to the subject, or at least the only recombinant agents that modulate
immune or inflammatory responses. In still other embodiments, the VLA-4
binding antibody alone is the only recombinant agent or the only biologic
administered to the subject.

[0243] All references and publications included herein are incorporated by
reference. The following examples are not intended to be limiting.

EXAMPLES

Example 1

[0244] Summary of development of intravenous formulation: storage and
structural stability. Experiments were performed to determine the storage
stability of a formulation of natalizumab containing 20 mg/mL natalizumab
in 10 mM phosphate, 140 mM sodium chloride, 0.02% polysorbate 80, pH 6.
Rates of stability change during storage of the formulation were
calculated for different storage condition data sets (2-8° C. for
24 months; 25° C. for 12 months; 40° C. for 3 months). The
following parameters were used to assess the rates of stability change
compared to the initial time point: percentage of aggregation, percentage
of oxidation of amino acid residue Methionine 255, percentage of
half-antibody present, percentage of H+L, percentage of fragmentation,
percentage of sialylation, and percentage of isoforms with a lower
isoelectric point (pI). The results of storage stability formulation
developmental trials are shown in Table 2. These results also provide a
baseline for stability studies of formulations containing higher antibody
concentrations.

[0245] Various biophysical techniques were used to assess the structural
stability of the 20 mg/mL natalizumab formulation, including monitoring
protein secondary structure through differential scanning calorimetry
(DSC), monitoring the environment of tryptophan residues through
tryptophan fluorescence with a fluorescent spectrophotometer, monitoring
the tertiary structure of the IgG fold via circular dichroism in the far
UV, and monitoring the environment of aromatic residues via UV and
visible spectrophotometry.

[0246] Structural stability of the 20 mg/mL formulation was optimized, in
part, by monitoring the Tm (midpoint of thermal melting curve)
during thermal melting experiments in a differential scanning calorimeter
(Microcal, Amherst, Mass.). Excess enthalpy needed to melt the protein
compared to buffer control was monitored and data were analyzed by
computer. Tryptophan fluorescence, known to be sensitive to structural
stability, was monitored by UV spectrophotometry in increasing
temperatures to optimize formulation conditions. Again, UV
spectrophotometry was used in the far UV region to measure the IgG fold.
Six peaks were monitored, and the second derivative of the UV-Vis
spectrum 230-320 nm was calculated in order to follow the environment of
the aromatic residues as a measure of structural stability in various
formulations.

[0247] Results of structural studies conducted to assess the stability of
natalizumab at 20 mg/mL are shown in Table 3. Additional studies have
shown that natalizumab undergoes heterogeneous nucleation during pumping
with piston pumps. No change in antibody quality was observed after
multiple freeze/thaw cycling and the antibody was stable after prolonged
2-3 days agitation in vials (data shown in Example 2). Natalizumab
undergoes aggregation, oxidation, and deamidation in the presence of
intense light stress and is also sensitive to metal-catalyzed oxidation
when stored in contact with stainless steel for long periods of time.

[0250] Accelerated stability tests were performed as described above were
also monitored by UV absorbance to detect aggregates. A UV
spectrophotometer was used to monitor the presence of aggregates pre- and
post-agitation by measuring absorbance at 340 nm for solutions of
natalizumab at a concentration of 150 mg/mL in HCN, HOL, PCN, and PST
buffers as described above, in a UV/Vis spectrophotometer with a 1 cm
path length.

[0251] The results of the UV absorbance study of aggregation of
natalizumab (150 mg/mL) in various formulation pre- and post-agitation
can be seen in FIG. 2. The histidine formulations (HOL and HCN) were most
stable under these conditions, as evidenced by the lower absorbance at
340 nm, indicating less aggregation. Insoluble precipitates were observed
in the phosphate formulations after storage for 1 month at 40° C.,
but were not observed in the histidine formulations.

[0253] Polysorbate-80 has a solubilizing effect on natalizumab. The
solubilizing effects of polysorbate-80 levels on natalizumab at 20 mg/mL
and 150 mg/mL were investigated. Polysorbate-80 was added at various
concentrations (w/v) to 20 mg/mL and 150 mg/mL natalizumab in HOL buffer.
Accelerated stability testing was performed by agitating the materials as
described above, for 8 weeks at 40° C. Percentage aggregation was
measured by SEC as described in the Example above.

[0254] The effect of polysorbate-80 levels on the stability of natalizumab
during agitation are shown in FIG. 3, which shows data for natalizumab at
135-165 mg/mL. The minimum polysorbate-80 level for stability during
agitation experiments is approximately 0.03% (w/v) for 150 mg/mL
natalizumab, and 0.02% (w/v) for 20 mg/mL natalizumab. Thus the preferred
polysorbate-80 concentration varies according to the antibody
concentration.

Example 4

[0255] Long term storage of natalizumab at different temperatures. The
stability of 150 mg/mL natalizumab in various formulations and at between
2-8° C. and at 40° C. was assessed by measuring the
percentage of aggregation using size exclusion chromatography as
described in Example 2 above. Natalizumab (150 mg/mL) was either in HCN,
HOL, PCN, or PST buffers as described in Example 2 above. Data for
natalizumab stored at 40° C. was collected over five months;
natalizumab stored at between 2-8° C. was collected over
twenty-four months.

[0256] The results of the stability effects of various formulations of
natalizumab (150 mg/mL) for storage at 40° C. are shown in FIG. 4;
results for 2-8° C. storage are shown in FIG. 5. At a
concentration of 150 mg/mL, natalizumab in the PST formulation (20 mM
phosphate, 140 mM NaC1, 0.02% (w/v) polysorbate 80, pH 6) aggregated the
least at 40° C. No significant differences in aggregation were
observed when the antibody was stored at 2-8° C. for twenty-four
months. These data demonstrated that the high-concentration of
natalizumab in the PST formulation is particularly stable.

Example 5

[0257] Methionine oxidation of natalizumab reduced in phosphate
formulations The tendency of methionine residues to oxidize in various
formulations of natalizumab (150 mg/ml), and the protective effects of
free methionine (10 mM) was studied by UV-HPLC characterization of
endoLysC peptide maps. Natalizumab (150 mg/mL) was stored at between
2-8° C. and at 40° C. for up to twenty-four months in the
histidine and phosphate formulations (HOL, HCN, PST, and PCN, as
described in Example 2). The results, expressed as percentage of oxidized
methionines, are shown in FIG. 6. For the phosphate formulations,
oxidation of methionines in natalizumab at 150 mg/mL was significantly
reduced and occurred at a lower rate in both temperature ranges, as
compared to the histidine formulations.

[0258] The anti-oxidant effects of excess free methionine (L-met) in the
histidine formulations, such as HOL, were seen when compared to
methionine-free formulations for natalizumab (150 mg/mL) stored at
40° C. over a time period of six months (FIG. 7). Percentage of
methionine oxidation was quantitated as described above. Thus,
formulating natalizumab at 150 mg/mL in phosphate buffers was more
protective against methionine oxidation, and reduced the number of
excipients required for stability at 150 mg/mL.

[0260] The results of fragmentation studies, which measured the percentage
of half antibodies over time for natalizumab at four different
concentrations (20 mg/mL, 50 mg/mL, 75 mg/mL, and 150 mg/mL) and in two
different formulations (HOL: 20 mM histidine, 240 mM glycerol, 0.04%
(w/v) polysorbate 80, pH 6; and PST: 20 mM phosphate, 140 mM NaC1, 0.02%
(w/v) polysorbate 80, pH 6) are shown in FIG. 8. The hinge cleavage rate
is lower in phosphate than in histidine formulations. The histidine
formulation environment is more reducing than the phosphate formulation
environment (based on redox potential).

Example 7

[0261] Exemplary formulations of natalizumab suitable for subcutaneous or
intramuscular administration. In view of the experiments above,
phosphate-based formulations were determined to be optimal for highly
concentrated natalizumab compositions suitable for SC or IM
administration. The compositions can also be diluted and used for IV
administration. The above experiments indicate that natalizumab is less
susceptible to oxidation when stored in phosphate formulations, such as
PST and PCN, than when stored in the histidine formulations HOL and HCN.
Therefore free methionine is typically not required in the phosphate
formulations to prevent oxidation of natalizumab (see Example 5). The
rate of fragmentation of natalizumab in the phosphate formulation PST was
also observed to be less than that in the histidine formulation HOL (see
Example 6). Long term stability was better in the phosphate formulations
PST and PCN than in the histidine formulations HOL and HCN.

[0262] In some embodiments, each of the components of the formulations
provided below can vary by 10%.

Example 8

[0263] Exemplary Formulation

[0264] 150 mg/mL Natalizumab

[0265] 10 mM sodium phosphate buffer

[0266] 140 mM sodium chloride

[0267] 0.04% (w/v) polysorbate 80 pH adjusted to pH 6.0±0.5

Example 9

[0268] Exemplary Formulation

[0269] 150 mg/mL Natalizumab

[0270] 10 mM sodium phosphate buffer

[0271] 275 mM glycerol

[0272] 0.04% (w/v) polysorbate 80

[0273] pH adjusted to pH 6.0±0.5

Example 10

[0274] Exemplary Formulation

[0275] 150 mg/mL Natalizumab

[0276] 10 mM sodium phosphate buffer

[0277] 160 mM L-arginine hydrochloride

[0278] 0.04% (w/v) polysorbate 80

[0279] pH adjusted to pH 6.0±0.5

Example 11

[0280] Exemplary Formulation

[0281] 150 mg/mL Natalizumab

[0282] 10 mM sodium phosphate buffer

[0283] 9% (w/v) sucrose

[0284] 0.04% (w/v) polysorbate 80

[0285] pH adjusted to pH 6.0±0.5

Example 12

[0286] Exemplary Formulation

[0287] 150 mg/mL Natalizumab

[0288] 10 mM sodium phosphate buffer

[0289] 9% (w/v) sorbitol

[0290] 0.04% (w/v) polysorbate 80

[0291] pH adjusted to pH 6.0±0.5

Example 13

[0292] Exemplary Formulation

[0293] 150 mg/mL Natalizumab

[0294] 20 mM L-histidine

[0295] 240 mM glycerol

[0296] 10 mM L-methionine

[0297] 0.04% (w/v) polysorbate 80

[0298] pH adjusted to pH 6.0±0.5

Example 14

[0299] Exemplary Formulation

[0300] 150 mg/mL Natalizumab

[0301] 20 mM L-histidine

[0302] 240 mM glycerol

[0303] 0.04% (w/v) polysorbate 80

[0304] pH adjusted to pH 6.0±0.5

Example 15

Stability Data

[0305] The formulation provided above as example 8 was stored in a staked
needle syringe, and stability data were measured at various time points.
These data are summarized in the Tables below and indicate that the
formulation, when stored in a syringe at 5° C., is stable for at
least 18 to 24 months.

[0316] A number of embodiments of the invention have been described.
Nevertheless, it will be understood that various modifications may be
made without departing from the spirit and scope of the invention.
Accordingly, other embodiments are within the scope of the following
claims.